Urinary tract infections are one of the most common and serious bacterial infections in a pediatric population. So far, they have mainly been related to age, gender, ethnicity, socioeconomic level, and the presence of underlying anatomical or functional, congenital, or acquired abnormalities. Recently, both innate and adaptive immunities and their interaction in the pathogenesis and the development of UTIs have been studied. The aim of this study was to assess the role and the effect of the two most frequent polymorphisms of TLR4 Asp299Gly and Thr399Ile on the development of UTIs in infants and children of Greek origin. We studied 51 infants and children with at least one episode of acute urinary tract infection and 109 healthy infants and children. We found that 27.5% of patients and 8.26% of healthy children carried the heterozygote genotype for TLR4 Asp299Gly. TLR4 Thr399Ile polymorphism was found to be higher in healthy children and lower in the patient group. No homozygosity for both studied polymorphisms was detected in our patients. In the group of healthy children, a homozygote genotype for TLR4 Asp299Gly (G/G) as well as for TLR4 Thr399Ile (T/T) was showed (1.84% and 0.92 respectively). These results indicate the role of TLR4 polymorphism as a genetic risk for the development of UTIs in infants and children of Greek origin.
Urinary tract infections (UTIs) are one of the most common and potentially important and serious bacterial infections in a pediatric population affecting approximately 2.6-7.5% of febrile children annually [
In the past, UTIs have mainly been related to age, gender, ethnicity, socioeconomic level, and the presence of underlying anatomical or functional, congenital, or acquired abnormalities. Recently, the function of innate immunity in the control of UTIs was studied. The need to clarify the role of the host (uroepithelium) response to recognize uropathogenic strains and the local immune response against their invasion has made the study of new risk factors imperative. The discovery of Pattern Recognition Receptors (PRRs) introduces the research of the role of innate immunity in relation to UTIs. PRRs are important immunologic biosensors that detect pathogens within the host’s cells and tissues by recognizing their structural components (PAMPS) [
Toll-like receptors (TLRs) are the best studied PRRs. Among them, 13 TLR family members recognize products of a variety of invading antigens. The TLR4 is a 224 amino acid protein that is encoded by the TLR4 gene in humans (
The two most studied SNPs of TLR4 in animal models and in vitro studies are TLR4 Asp299Gly and TLR4 Thr399Ile. Specifically, in the Asp299Gly polymorphism, we have an A to G transition (SNP
This is a case-control study in a population of Greek origin. In total, 160 infants and children were included. We studied 51 infants and children (19 males, 32 females) with at least one episode of acute urinary tract infection (AUTI) in their records, with or without underlying anatomic genitourinary anomalies (average age at entry to the study is
All patients who participated in the study should meet the following criteria: (a) Those suffering from lower urinary tract infection (cystitis) should have significant bacteriuria of a single microorganism (105 CFU mL-1), erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) at normal levels (
Patients formed Group A which was subsequently divided in two subgroups: Group A1 (35/51 patients)—infants and children diagnosed with lower UTI, and Group A2 (16/51 patients)—infants and children diagnosed with upper UTI. All patients were hospitalized at the Pediatric Clinic, Fourth Department of Pediatrics, Faculty of Medicine, Papageorgiou General Hospital, Aristotle University of Thessaloniki. The control group (Group C) consisted of healthy infants and children who were followed up at the General Pediatrics Outpatient Unit of the Fourth Department of Pediatrics.
In all patients and 79/109 of Group C, personal history was recorded, a detailed clinical examination was conducted, and a blood sample was collected for the determination of biochemical and immunological parameters as well as a urine sample. They had complete blood count, measurement of urea and creatinine, ESR, CRP, urine culture, and urine microbiological analysis. A complete immunological profile was also evaluated in every child including immunoglobulins: IgG IgA, IgM, IgG subclasses (IgG1, IgG2, IgG3, IgG4), and complement components C3 and C4. In all patients, kidney ultrasound was performed. In all 160 children of the study, genetic testing was performed to determine the TLR4 Asp299Gly and TLR4 Thr399Ile polymorphisms. Before entering the study, an informed consent was obtained by parents or guardians of the children. The research protocol was declared at the service of ClinicalTrials.gov and approved by the Ethics Committee of the Faculty of Medicine of Aristotle University of Thessaloniki. The study was conducted according to the criteria of the Declaration of Helsinki.
Whole blood was collected into a sterile vacutainer containing anticoagulant ethylenediaminetetraacetic acid (EDTA). Molecular genetic analysis was carried out on genomic DNA extracted from EDTA using according to the manufacturer’s instructions.
Genomic DNA was extracted from the whole blood using Invisorb Spin Blood Mini Kit (STRATEC Molecular GmbH, Germany) following the manufacturer’s instructions. The 249 bp gene fragment carrying the TLR4 Asp299Gly mutation was amplified from whole genomic DNA using the 5
The presence of TLR4 gene mutations was determined by RFLP. The 249 bp PCR product was cut with NcoI to determine the presence of TLR4 Asp299Gly mutation, and the 407 bp PCR product was cut with HinfI to determine for the presence of Thr399Ile mutation, as described by Lorenz et al. [
Subjects were subjected to electrophoresis on gels containing a mixture of 3% and 3.5% agarose gel.
Data were analyzed using the IBM statistical program Statistical Package for Social Science software for Windows, version 22.0 (SPSS Inc., Chicago, Illinois, USA). Expected and observed frequencies of genotypes and alleles in patients and controls were compared in
The demographic and the clinical features of Group A (patients), Subgroups A1 and A2 (patients), and Group C (controls) are shown in Tables
Demographic data of the children of the study.
Group Α (patients) | Group Α1 | Group Α2 | Group C (controls) | |
---|---|---|---|---|
51 | 35 | 16 | 109 | |
Age (years) Mean ± SD | ||||
0.001 | ||||
Males | 19 | 17 | 2 | 55 |
Females | 32 | 18 | 14 | 54 |
0.017 |
Clinical data of the patients.
Group A (patients) | Gender (M/F) | ||
---|---|---|---|
Total ( |
Females ( |
Males ( | |
Lower UTI (Group Α1) | 35 | 18 | 17 |
Upper UTI (Group Α2) | 16 | 14 | 2 |
1 episode of UTI | 41 | 28 | 13 |
≥2 episodes of UTI | 10 | 4 | 6 |
Gram-/Gram+ | 51/0 | 32/0 | 19/0 |
US (+/-) | 13/38 | 7/25 | 6/13 |
UTI: urinary tract infection; US: ultrasound.
Urine cultures of all patients showed Gram-negative bacteria with
Laboratory data are shown in Table
Laboratory data of the study groups.
Parameters | Patients ( |
Controls ( |
|
---|---|---|---|
Leukocytes (×103/u) | <0.001 | ||
Neutrophils (%) | <0.001 | ||
Lymphocytes (%) | <0.001 | ||
Absolute neutrophil count | <0.001 | ||
IgG (g/L) | <0.001 | ||
IgA (g/L) | <0.001 | ||
IgM (g/L) | 0.419 | ||
IgG1 (g/L) | <0.001 | ||
IgG2 (g/L) | <0.001 | ||
IgG3 (g/L) | <0.001 | ||
IgG4 (g/L) | <0.001 | ||
C3 (g/L) | <0.001 | ||
C4 (g/L) | 0.004 | ||
CRP (mg/dL) | <0.001 | ||
ESR (mm) | <0.001 | ||
Serum urea (mg/dL) | <0.001 | ||
Serum creatinine (mg/dL) | <0.001 |
In this study, the distribution of genotypes in both patients and healthy subjects did not differ significantly from the expected Hardy-Weinberg equilibrium (
The distribution of TLR4 Asp299Gly and Thr399Ile polymorphisms in patients and controls.
Patients | Controls | |||
---|---|---|---|---|
Frequency (%) | ||||
Group A ( |
Group A1 ( |
Group A2 ( |
Group C ( | |
TLR4 Asp299Gly | Genotype | |||
AA | 37 (72.5) | 25 (71.4) | 12 (75.0) | 98 (89.9) |
AG | 14 (27.5) | 10 (28.6) | 4 (25.0) | 9 (8.26) |
GG | 0 (0.00) | 0 (0.00) | 0 (0.00) | 2 (1.84) |
0.02446 | ||||
A | 88 (0.86) | 60 (0.86) | 28 (0.88) | 205 (0.94) |
G | 14 (0.14) | 10 (0.14) | 4 (0.12) | 13 (0.06) |
0.06361 | ||||
TLR4 Thr399Ile | Genotype | |||
CC | 50 (98.04) | 34 (97.1) | 16 (100) | 96 (88.07) |
CT | 1 (1.96) | 1 (2.9) | 0 (0.00) | 12 (11.01) |
TT | 0 (0.00) | 0 (0.00) | 0 (0.00) | 1 (0.92) |
0.3465 | ||||
C | 101 (0.99) | 69 (0.99) | 32 (1) | (0.94) |
T | 1 (0.01) | 1 (0.01) | 0 (0.00) | (0.06) |
0.542 |
The distribution of TLR4 Asp299Gly and TLR4 Thr399Ile genotypes carrying the predisposing allele did not differ significantly between males and females in the patient group as well as compared with the control group (Table
The distribution of TLR4 Asp299Gly and TLR4 Thr399Ile polymorphisms in patients and controls according to gender.
Gender | |||||||||
---|---|---|---|---|---|---|---|---|---|
SNPs | Genotype | Group Α ( |
Group Α1 ( |
Group Α2 ( |
Group C ( | ||||
Males | Females | Males | Females | Males | Females | Males | Females | ||
TLR4 Asp299Gly | AA | 15 (79) | 22 (69) | 14 (82) | 12 (67) | 1 (50) | 10 (71) | 50 (90) | 48 (89) |
4 (21) | 10 (31) | 3 (18) | 6 (33) | 1 (50) | 4 (29) | 5 (10) | 6 (11) | ||
0.4301 | 0.2886 | 0.5408 | 0.7263 | ||||||
TLR4 Thr399Ile | CC | 18 (95) | 32 (100) | 16 (94) | 18 (100) | 2 (100) | 14 (100) | 51 (93) | 45 (83) |
1 (5) | 0 | 1 (6) | 0 | 0 | 0 | 4 (7) | 9 (17) | ||
0.19 | 0.2965 | NA | 0.1303 |
Analysis of the relation between genotypes and the number of urinary tract infections and findings of the ultrasound.
Number of episodes | |||||||
SNPs | Genotypes | Group Α ( |
Group Α1 ( |
Group Α2 ( | |||
1 | ≥2 | 1 | ≥2 | 1 | ≥2 | ||
TLR4 Asp299Gly | AA | 29 (70.7) | 7 (70) | 19 (73) | 6 (66.7) | 11 (73.3) | 2 (100) |
12 (29.3) | 3 (30) | 7 (27) | 3 (33.3) | 4 (26.7) | 0 (0) | ||
0.9637 | 0.713 | 0.4036 | |||||
TLR4 Thr399Ile | CC | 40 (97.6) | 10 (100) | 25 (96.2) | 9 (100) | 15 (100) | 1 (100) |
1 (2.4) | 0 (0) | 1 (3.8) | 0 (0) | 0 (0) | 0 (0) | ||
0.6179 | 0.5505 | NA | |||||
Findings of the ultrasound | |||||||
SNPs | Genotypes | Group Α ( |
Group Α1 ( |
Group Α2 ( | |||
Normal (%) | Pathological (%) | Normal (%) | Pathological (%) | Normal (%) | Pathological (%) | ||
TLR4 Asp299Gly | AA | 27 (71) | 10 (77) | 19 (73) | 6 (66.7) | 8 (66.7) | 4 (100) |
11 (29) | 3 (23) | 7 (27) | 3 (33.3) | 4 (33.3) | 0 (0) | ||
0.4961 | 0.7137 | 0.1824 | |||||
TLR4 Thr399Ile | CC | 37 (97.3) | 13 (100) | 25 (96.1) | 8 (100) | 12 (100) | 4 (100) |
CT+TT | 1 (2.7) | 0 (0) | 1 (3.9) | 0 (0) | 0 (0) | 0 (0) | |
0.5547 | 0.5734 | NA |
Although 13/51 (25.5%) of the examined patients had abnormal kidney ultrasound findings, only 3 patients heterozygous for the TLR4 Asp299Gly polymorphism were among them. Therefore, no statistically significant association of the TLR4 Asp299Gly polymorphism with abnormal findings in kidney ultrasound was found (Table
Analysis of the relation between genotypes and WBC, neutrophils, lymphocytes, ESR, CRP, serum urea, and serum creatinine.
SNPs | Genotypes | |||||||
---|---|---|---|---|---|---|---|---|
WBC | Neutrophils (%) | Lymphocytes (%) | ESR (mm) | CRP (mg/dL) | Serum urea (mg/dL) | Serum creatinine (mg/dL) | ||
TLR4 Asp299Gly | AA | |||||||
0.286 |
0.506 |
0.540 |
0.165 |
0.950 |
0.325 |
0.212 | ||
TLR4 Thr399Ile | CC | |||||||
11720 | 34.3 | 56.3 | - | 0.39 | 23 | 0.53 | ||
0.634 |
0.154 |
0.135 |
N/A | 0.248 |
0.785 |
0.377 |
Analysis of the relation between genotypes and immunoglobulins, IgG subclasses, C3, and C4.
SNPs | Genotypes | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
IgG (g/L) | IgA (g/L) | IgM (g/L) | IgG1 (g/L) | IgG2 (g/L) | IgG3 (g/L) | IgG4 (g/L) | C3 (g/L) | C4 (g/L) | ||
TLR4 Asp299Gly | AA | |||||||||
0.347 |
0.101 |
0.709 |
0.634 |
0.542 |
0.277 |
0.283 |
0.488 |
0.888 | ||
TLR4 Thr399Ile | CC | |||||||||
5.210 | 0.530 | 1.050 | 4.020 | 0.860 | 0.340 | 0.180 | 1.540 | 0.329 | ||
0.572 |
0.944 |
0.572 |
0.735 |
0.735 |
0.598 |
0.970 |
0.590 |
0.190 |
Urinary tract infection is one of the most usual bacterial infections in infancy and childhood that occurs in 8.4% of girls and 1.7% of boys under the age of 6 years [
The urinary tract is continuously exposed to microorganisms and pathogens mainly coming from the intestinal tract. Ιt resists by developing defense mechanisms that represent mainly functions of innate immunity. Uroepithelium arises barriers, especially when it encounters infections of the lower urinary tract. For this reason, the role of innate immunity is the best studied regarding the pathogenesis of urinary tract infections compared to that of adaptive immunity.
Over the last 20 years, researchers have studied both innate and adaptive immunities and their interaction in the pathogenesis and the development of UTIs. It is suggested that genetic factors are capable of interfering with any step during the process of bacteria invasion and regulate the inflammatory response before, during, and after UTI. These factors are different gene products such as cytokines, receptors, and adhesion molecules. TLRs are receptors expressed either on the surface of many cells in the blood or on the surface of epithelial cells, such as uroepithelium. They play a pivotal role in the identification of infectious factors and the rapid activation of signaling pathways for the elimination of microbial pathogens or the mobilization of adaptive immunity. It is perceived that TLRs are acting as a connecting bridge between innate and adaptive immunities. In the last 10 years, researchers have focused on polymorphisms or polymorphism combinations of genes coding for TLRs [
The aim of this study was to assess the role and the effect of the two most frequent polymorphisms of TLR4 Asp299Gly and Thr399Ile on the development of UTIs in infants and children of Greek origin. Identifying the possible role of genetic variants in UTIs would help clinicians understand the pathogenesis of the inflammation and distinguish which children have a greater risk of developing UTI, based on their genetics.
The association of TLR4 Asp299Gly or Thr399Ile polymorphisms with a faulty response to the lipopolysaccharide (LPS) of Gram-negative bacteria in mice was first reported by Arbour et al. in 2000. They suggested that these mutations of the TLR gene may affect the TLR structure or expression and therefore have a negative effect on the response to bacterial endotoxins. This was later confirmed by many other studies [
Case-control studies, so far, have established associations of TLR4 Asp299Gly or Thr399Ile polymorphisms with the development of a variety of diseases. Most of the studies were conducted on adult populations.
The first reference to the relationship between TLR4 and UTIs is found in 2003 by Schilling et al., who demonstrated that TLR4 on stromal and hematopoietic cells, in mice, mediates innate resistance to uropathogenic
Since 2006, however, numerous and continuously growing studies on human populations have been done dealing with TLR4 polymorphisms and their association with UTIs [
Reviewing the literature, we found that the first to study a possible relation of TLR4 polymorphisms with UTIs were Karoly et al. [
In our results, we found that 27.5% of patients and 8026% of healthy children carried the heterozygote genotype for TLR4 Asp299Gly, a difference which was statistically significant. On the opposite, TLR4 Thr399Ile polymorphism was higher in healthy children (11.01%) and lower in the patient group (1.96%). No homozygosity for both studied polymorphisms was detected in our patients. However, in the group of healthy children in our study, 1.84% showed a homozygote genotype for TLR4 Asp299Gly (G/G) and 0.92% showed a homozygote genotype for TLR4 Thr399Ile (T/T).
Mutlubas et al. [
In this study, we made an attempt to record the frequency of TLR4 Asp299Gly and TLR4 Thr399Ile polymorphisms in healthy children and in children with one or more episodes of UTI. Moreover, we tried to look into a possible relation between the presence of these polymorphisms with parameters from their personal history such as gender, number of UTI episodes, renal function, kidney ultrasound findings, and laboratory data. What differentiates our study is that the percentage of the heterozygous for the TLR4 Asp299Gly polymorphism patients was much higher than any other percentage recorded in similar studies so far. TLR4 Thr399Ile polymorphism was also found to be more frequent in healthy children. Our results suggest a causative role of the TLR4 Asp299Gly polymorphism in the occurrence and progression of UTIs in children of Greek origin while, on the contrary, a protective role of the TLR4 Thr399Ile polymorphism against urinary tract infections. These findings could be attributed to the fact that the distribution of many SNPs differs among different populations. It should also be taken into consideration the fact that these results may be applicable in the Greek children and not in the general population. More and larger studies need to be conducted to confirm this statement.
Our data is the first to be published in Greece, addressing the role of TLR4 Asp299Gly and TLR4 Thr399Gly polymorphisms as a genetic risk for the development of UTIs in infants and children of Greek origin. We identify the possible role of the TLR4 genetic variants in the pathogenesis of UTI that would help to early recognition of the children at a greater risk and could serve as a useful tool to personalized management of UTIs in children. Our study contributes to growing evidence of the role of TLRs to UTI, however indicating the need for a larger sample and more multicenter studies.
The data used to support the findings of this study are available from the corresponding author upon request.
This research was performed as part of the employment of the authors.
No conflict of interests has been declared by the authors.